Rev1, Rev3, or Rev7 siRNA Abolishes Ultraviolet Light-Induced Translesion Replication in HeLa Cells: A Comprehensive Study Using Alkaline Sucrose Density Gradient Sedimentation
Figure 4
Efficient knockdown by Polη siRNAs and the effects on UV-induced TLS in HeLa cells (ASDG profiles of replication products). (a) Dose-response of siPolη-4 on UV-TLS, Efficiency of knockdown on Polη expression; (b) RT-PCR analysis and western blot analysis, (c) Effects of various Polη siRNAs on UV-TLS, (d) Effect of various Polη siRNAs (no UV control). Twenty-four hours after Polη siRNA transfection, total RNA was isolated and Polη RNA was quantified by RT-PCR. Results were shown in MultiNA gel images and the expression level was presented under the panel (b). Forty hours after Polη siRNA transfection, whole cell extracts were prepared and Polη protein was quantified by western blot analysis (b). Forty hours after Polη siRNA transfection, cells were UV-irradiated (10 J/m2), incubated in normal medium for 30 minutes, pulse-labelled with 10 μCi/mL of [14C]thymidine for 1 hour, then washed twice with PBS, and incubated for 5 hours at 37°C in normal medium (a, c). Forty hours after Polη siRNA transfection, cells were not UV-irradiated, pulse-labelled with 10 μCi/mL of [14C]thymidine for 30 minutes, washed twice with PBS, and incubated at 37°C in normal medium for 1 hour (d). Some of these profiles overlap (d). Sedimentation is from right to left. The arrow indicates the position of T4 phage DNA (166 kb, i.e., approximately 5.5 × 107 Da/single strand). Average fragment length (in Mb) of each profile is shown in square brackets.
